Method of determining ligand

ABSTRACT

The present invention aims at providing a method of determining a ligand to an orphan receptor. Specifically, the present invention provides a method of determining a ligand to a receptor protein, to which no ligand has been determined, which comprises using a fusion protein of the receptor protein and a fluorescent protein.

FIELD OF THE INVENTION

The present invention relates to a method of determining a ligand to areceptor protein present on cell membrane and in particular, relates toa method of determining a ligand to a so-called orphan receptor, ofwhich the ligand is totally unknown.

BACKGROUND ART

Physiologically active substances such as various hormones andneurotransmitters regulate the biological function through specificreceptor proteins present on cell membranes. Many of these receptorproteins are coupled with guanine nucleotide-binding protein(hereinafter sometimes simply referred to as G protein) and mediate theintracellular signal transduction through activation of G protein. Thesereceptor proteins have the common structure containing seventransmembrane domains and are thus collectively referred to as Gprotein-coupled receptor proteins or seven-transmembrane receptorproteins (7TMR).

G protein-coupled receptor proteins are present on the cell surface ofeach functional cell and organ in an organism, and play importantphysiological roles as the target of the molecules that regulate thefunctions of the cells and organs, e.g., hormones, neurotransmitters,physiologically active substances or the like. Receptors transmitsignals to cells through binding with physiologically active substances,and the signals induce various reactions such as activation orsuppression of the cells.

To clarify the relationship between substances that regulate thefunctions of cells and organs in various organisms, and their specificreceptor proteins, in particular, G protein-coupled receptor proteinswould elucidate the functions of cells and organs in various organismsto provide a very important means for development of pharmaceuticalsclosely associated with the functions.

Substances that inhibit binding of G protein-coupled receptor proteinsto physiologically active substances (i.e., ligands) or substances thatbind and induce signal transduction similar to that induced byphysiologically active substances (i.e., ligands) have been hithertoused for pharmaceuticals, as antagonists and agonists specific to thesereceptors that regulate the biological functions.

However, even at the present moment, there are a large number ofso-called orphan receptors in which the corresponding ligands are yetunidentified. Thus, it has been earnestly desired to identify theligands and elucidate their functions.

G protein-coupled receptors are useful in searching for a novelphysiological active substance (i.e., ligand) using the signaltransduction activity as the index and in search for agonists andantagonists to the receptor. Ligands, agonists, antagonists, or the liketo these receptors are expected to be used as prophylactic and/ortherapeutic and diagnostic agents for diseases associated withdysfunction of the G protein-coupled receptors.

GFP (Green Fluorescent Protein) is one of fluorescent proteins derivedfrom the jellyfish Aequorea Victoria of mesozoan animals (WO 96/23810,WO 96/27675, WO 97/26333, WO 97/28261, WO 97/42320).

A method of determining a ligand to Gi-, Gs- or Gq-coupled orphanreceptor using multiple response elements (MRE) and a cAMP responseelement (CRE) in combination with a reporter activity is reported(Analytical Biochemistry, 275, 54-61, 1999).

A method of screening molecules which interact on Gs- or Gq-coupledorphan receptors using a cAMP response element (CRE) in combination witha reporter activity is reported (Analytical Biochemistry, 226, 349-354,1995).

A method of characterization for Gi- or Gs-coupled receptors with knownligands using a cAMP response element (CRE) in combination with areporter activity is reported (Current Opinion in Biotechnology, 1995,6: 574-581).

A method of detecting the response of a G protein-coupled receptor usinga cell in which chimera G protein a subunits with increased promiscuityin coupling specificity for the G protein-coupled receptor, chimera Gprotein has been expressed is reported (WO 01/36481).

A method of identifying a ligand for an orphan G protein-coupledreceptor using a recombinant yeast expression library is reported (U.S.Pat. No. 6,255,059).

A method of identifying a receptor activity modifying substance composedof a cell containing both a receptor and a test peptide, which cell isuseful for identifying or screening an orphan receptor is reported (US2001/0026926).

An expression system for identifying a ligand to an orphan receptorutilizing the gamogenesis system of Saccharomyces is reported (WO00/031261).

In conventional methods for searching/identifying a ligand, for example,when a ligand is screened using an eukaryotic cell, a so-called stablecell line capable of stably expressing its receptor should beestablished, and a special cell was required for establishing the cellline. Moreover, it was necessary for the screening to use a plurality ofassays in combination. Thus, when a plurality of test compounds existed,it took a long time, which made it difficult to assay them. That is,conventional methods for searching/identifying ligands, etc. encounterproblems that (1) a usable cell line is restricted, (2) it takes time toestablish the cell line, (3) because of using a plurality of assays incombination, the number of specimens increases so that it becomesdifficult to implement the methods, and so on. A method of determining aligand that solves these problems and can use various cell lines andimplement in a short period of time is desired.

DISCLOSURE OF THE INVENTION

The present inventors made extensive investigations. As a result, bypreparing a cell wherein a fusion protein of a receptor protein, towhich no ligand has been determined, and a fluorescent protein such asGFP was stably or transiently expressed, the inventors have found (1) to(5) below, using immunostaining assay, western blotting assay, etc.utilizing fluorescence from a fluorescent protein such as GFP or afluorescent protein antibody such as a GFP antibody that:

-   -   (1) expression of the receptor protein could be confirmed on a        protein level;    -   (2) expression of the receptor protein on a cell membrane could        be confirmed;    -   (3) an expression level of the receptor protein could be        approximated;    -   (4) a cell with the receptor protein being highly expressed        could be screened; and,    -   (5) a specific reaction of the receptor with the ligand could be        detected as intracellular internalization of the fusion protein        of the receptor and the fluorescent protein; etc. By utilizing        these characteristics, the inventors found that a ligand to the        receptor protein for which a ligand has not been identified        (hereinafter sometimes simply referred to as an orphan receptor)        could efficiently be determined. Based on these findings, the        present inventors have made further investigations and come to        accomplish the present invention.

That is, the present invention relates to the following features:

-   -   [1] A method of determining a ligand to a receptor protein for        which a ligand has not been identified, which comprises using a        fusion protein of the receptor protein with a fluorescent        protein;    -   [2] The ligand determination method according to [1], wherein        the fusion protein of a receptor protein for which a ligand has        not been identified and GFP is used;    -   [3] The ligand determination method according to [1], wherein a        cell expressed with the fusion protein of a receptor protein for        which a ligand has not been identified and GFP or a membrane        fraction of the cell;    -   [4] The ligand determination method according to [1], which        comprises assaying (1) an activity that accelerates or        suppresses arachidonic acid release, acetylcholine release,        intracellular Ca²⁺ release, intracellular cAMP production,        intracellular cGMP production, inositol phosphate production,        changes in cell membrane potential, phosphorylation of        intracellular proteins, activation of c-fos or pH reduction, (2)        MAP kinase activation, (3) transcription factor activation, (4)        diacylglycerol production, (5) opening or closing of ion        channels on a cell membrane, (6) apoptosis induction, (7)        morphological changes in cells, (8) transport of the fusion        protein from cell membrane to cytoplasm, (9) low molecular        weight G protein activation, (10) cell divisioh-promoting        activity or (11) DNA synthesis-promoting activity;    -   [5] The ligand determination method according to [1], wherein        transport of the fusion protein from cell membrane to cytoplasm        is assayed;    -   [6] The ligand determination method according to [5], wherein        transport of the fusion protein from cell membrane to cytoplasm        is assayed by observing the fluorescence of GFP;    -   [7] The ligand determination method according to [1], which        comprises bringing a cell capable of expressing the fusion        protein of a receptor protein for which a ligand has not been        identified the receptor protein and a fluorescent protein and        containing a plasmid ligated with a DNA encoding a reporter        protein at the downstream of cAMP response element/promoter in        contact with a test compound and assaying the activity of the        reporter protein;    -   [8] The method according to [7], which comprises culturing a        cell containing (1) a plasmid containing a DNA encoding the        fusion protein of a receptor protein for which a ligand has not        been identified and a fluorescent protein and (2) a plasmid        ligated with a DNA encoding a reporter protein at the downstream        of cAMP response element/promoter, bringing the cell in contact        with a test compound and assaying the activity of the reporter        protein;    -   [9] The ligand determination method according to [2], wherein a        cell capable of expressing the fusion protein of a receptor        protein for which a ligand has not been identified and GFP and        containing a plasmid ligated with a DNA encoding a reporter        protein at the downstream of cAMP response element/promoter and        assaying the activity of the reporter protein;    -   [10] The ligand determination method according to [9], which        comprises culturing a cell containing (1) a plasmid containing a        DNA encoding the fusion protein of a receptor protein for which        a ligand has not been identified and GFP and (2) a plasmid        ligated with a DNA encoding a reporter protein at the downstream        of cAMP response element/promoter, bringing the cell in contact        with a test compound and assaying the activity of the reporter        protein;    -   [11] The method according to [1], wherein the receptor protein        is a G protein-coupled receptor protein;    -   [12] The method according to [1], wherein GFP is a protein        containing the same or substantially the same amino acid        sequence as the amino acid sequence represented by SEQ ID NO: 1,        SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7;    -   [13] The method according to [7], wherein the promoter is a        TATA-like sequence;    -   [14] The method according to [7], wherein the reporter protein        is luciferase;    -   [15] The method according to [7], wherein the plasmid is a        plasmid ligated with a TATA-like promoter and a gene encoding        the reporter protein at the downstream of cAMP response element;    -   [16] The method according to [7], wherein the cell has expressed        at least two receptor proteins for which ligand s have not been        identified;    -   [17] The method according to [7], wherein the cell further        contains a plasmid containing a gene encoding an inhibitory G        protein α subunit Gi;    -   [18] The method according to [7], wherein forskolin is further        added;    -   [19] The method according to [16], wherein the at least two        receptor proteins have similar characteristics;    -   [20] The method according to [19], wherein the similar        characteristics are the basic expression level of the reporter        protein and/or the expression level of the reporter protein when        forskolin is added;    -   [21] The method according to [16], which comprises measuring the        basic expression level of the reporter protein when the at least        two receptor proteins are previously expressed individually        and/or the expression level of the reporter protein by the        addition of forskolin and being expressed in combination the at        least two receptor proteins having an equivalent expression        level of the reporter protein;    -   [22] A fusion protein of a receptor protein for which a ligand        has not been identified and a fluorescent protein, or a salt        thereof;    -   [23] The fluorescent protein or a salt thereof according to        [22], wherein the fluorescent protein is GFP;    -   [24] A DNA containing a DNA encoding the fusion protein        according to [22];    -   [25] A recombinant vector containing the DNA according to [22];    -   [26] A transformant transformed with the recombinant vector        according to [25];    -   [27] Use of a fluorescent protein to determine a ligand to a        receptor protein for which a ligand has not been identified;        and,    -   [28] Use of GFP to determine a ligand to a receptor protein for        which a ligand has not been identified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results (n=2) of detection of reporter activation bycholesterol metabolism-related substances in HEK293 cells transfectedwith TGR5 expression vector (A) and HEK293 cells transfected with intactvector alone (B).

FIG. 2 shows the results of induced expression of reporter genes byligand stimulation in HEK293 cells.

FIG. 3 shows the results of TGR5 response to lithocholic acid in theco-presence of Gi.

FIG. 4 shows localization of the TGR5-GFP fusion protein expressed inCHO cells in the absence of ligand. In the figure, white line shows thelength of 4 μm.

FIG. 5 shows localization of the fusion protein 30 minutes after theaddition of TLCA to TGR5-GFP-expressed CHO cells. In the figure, whiteline shows the length of 4 μm.

BEST MODE FOR CARRYING OUT THE INVENTION

The ligand determination method of the present invention ischaracterized by using (1) a fusion protein of a receptor protein forwhich a ligand has not been identified and a fluorescent protein, morespecifically, is a method which comprises bringing a cell wherein thefusion protein of a receptor protein for which a ligand has not beenidentified and a fluorescent protein has been expressed or a membranefraction of the cell in contact with a test compound (liganddetermination method A).

Furthermore, the ligand determination method of the present inventionincludes (1) a method which involves bringing a cell capable ofexpressing a fusion protein of a receptor protein for which a ligand hasnot been identified and a fluorescent protein and containing a plasmidligated with DNA encoding a reporter protein at the downstream ofenhancer/promoter in contact with a test compound and assaying theactivity of the expression-induced reporter protein, and (2) a methodwhich involves culturing a cell containing (i) a plasmid containing DNAencoding a fusion protein of a receptor protein for which a ligand hasnot been identified and a fluorescent protein and (ii) a plasmid ligatedwith DNA encoding a reporter protein at the downstream ofenhancer/promoter, and bringing the cell in contact with a test compoundand assaying the activity of the expression-induced reporter protein(ligand determination method B).

As the orphan receptors used in the present invention, for example, Gprotein-coupled receptor proteins, etc. are employed. Specific examplesinclude G protein-coupled receptor proteins containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 9, G protein-coupled receptor proteinsdescribed in WO 96/05302, EP-A-711831, EP-A-789076, EP-A-1103563,EP-A-1103562, Published Japanese Patent Application KOKAI Nos. 8-154682,8-283295, 8-196278, 8-245697, 8-266280, 9-51795, 9-121865, 9-2388686 and10-146192, and the like.

The fluorescent proteins are not particularly limited as far as it isvisually recognizable, and include, e.g., GFP (Green FluorescentProtein), Tag sequence, EGFP (enhanced green fluorescent protein), ECFP(enhanced cyan fluorescent protein), EYFP (enhanced yellow fluorescentprotein), DsRED (Discosoma sp. red fluorescent protein), EBFP (enhancedblue fluorescent protein), etc.

GFP is one of fluorescent proteins derived from the jellyfish AequoreaVictoria of mesozoan animals, and includes proteins containing the sameor substantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,etc.

The amino acid sequence which is substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 includes an amino acid sequencehaving at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7;or the like.

Preferred examples of the protein of the present invention whichcontains substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQID NO: 7 include a protein having substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 and having the activitysubstantially equivalent to proteins comprising the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7;and the like.

The activities of GFP include, e.g., fluorescence by excitation lightirradiation, etc. The term substantially equivalent activities are usedto mean that these activities are equivalent in nature. Thus, though itis preferred that these activities are equivalent (e.g., about 0.01- toabout 100-fold, preferably about 0.5- to about 20-fold, more preferablyabout 0.5- to about 2-fold), quantitative factors such as an activitylevel, a molecular weight of the protein, etc. may be different.

The activities in GFP such as fluorescence by excitation lightirradiation, etc. can be assayed by publicly known methods withmodifications.

Proteins containing the following amino acid sequences are used as GFP:(1) the amino acid sequences wherein at least 1 or 2 amino acids(preferably approximately 1 to 30 amino acids, more preferablyapproximately 1 to 10 amino acids, most preferably several (1 to 5)amino acids) are deleted in the amino acid sequence represented by SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; (2) the amino acidsequences wherein at least 1 or 2 amino acids (preferably approximately1 to 30 amino acids, more preferably approximately 1 to 10 amino acids,and most preferably several (1 to 5) amino acids) are added to orinserted into the amino acid sequence represented by SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; (3) the amino acid sequenceswherein at least 1 or 2 amino acids (preferably approximately 1 to 30amino acids, more preferably approximately 1 to 10 amino acids, and mostpreferably several (1 to 5) amino acids) are substituted by other aminoacids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 or SEQ ID NO: 7; or (4) the amino acid sequence whereinthese deletion, addition and substitution are combined; etc. Amongothers, preferably used are proteins containing (i) the amino acidsequence wherein the N-terminal methionine residue is deleted in theamino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5 or SEQ ID NO: 7, (ii) the amino acid sequence wherein theN-terminal methionine residue is deleted and the following alanineresidue is substituted with a threonine residue or serine residue in theamino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5 or SEQ ID NO: 7; etc.

Specifically, there are used, for example, (i) GFP consisting of aminoacid sequences represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7, (ii) GFP consisting of amino acid sequences representedby SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, wherein theN-terminal methionine residue is deleted, (iii) GFP consisting of aminoacid sequences represented by SEQ ID NO: 1, wherein the N-terminalmethionine residue is deleted and the following alanine residue issubstituted with a threonine residue or serine residue; etc.

As the Tag sequence, for example, known sequences below are employed.(SEQ ID NO: 17) (1) His-tag (PCDNA3.1/His A) His His His His His His(SEQ ID NO: 18) (2) V5-tag (PCDNA3.1/V5-His A) Gly Lys Pro Ile Pro AsnPro Leu Leu Gly Leu Asp Ser Thr (SEQ ID NO: 19) (3) myc-tag(pCDNA3.1/myc-His A) Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu (SEQ ID NO:20) (4) Xpress-tag (PCDNA3.1/His A) Asp Leu Tyr Asp Asp Asp Asp Lys (SEQID NO: 21) (5) HA-tag (PCluzHA Expression vector) Met Gly Ser Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser Leu Glu Phe

As EGFP, there is used a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 7.

As ECFP, there is used a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 22.

As EYFP, there is used a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 24.

As DsRtD, there is used a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 26.

As EBFP, there is used a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 28.

These EGFP, ECFP, EYFP, DsRED and EBFP may be proteins having (1) theamino acid sequence wherein at least 1 or 2 amino acids (preferablyapproximately 1 to 30 amino acids, more preferably approximately 1 to 10amino acids, most preferably several (1 to 5) amino acids) are deletedin the amino acid sequences described above; (2) the amino acid sequencewherein at least 1 or 2 amino acids (preferably approximately 1 to 30amino acids, more preferably approximately 1 to 10 amino acids, and mostpreferably several (1 to 5) amino acids) are added to or inserted intothe amino acid sequences described above; (3) the amino acid sequencewherein at least 1 or 2 amino acids (preferably approximately 1 to 30amino acids, more preferably approximately 1 to 10 amino acids, and mostpreferably several (1 to 5) amino acids) are substituted by other aminoacids in the amino acid sequences described above; or (4) the amino acidsequence wherein these deletion, addition and substitution are combined;etc., so long as they have the activities such as fluorescence byexcitation light irradiation, etc.

Specifically, the DNA encoding GFP may be any DNA, so long as it is, forexample, DNA containing the base sequence represented by SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or DNA having a basesequence hybridizable to a complementary base sequence to the basesequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQID NO: 8 under high stringent conditions and encoding a protein whichhas the activities (e.g., fluorescence by excitement light irradiation,etc.) substantially equivalent to those of GFP consisting of the aminoacid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 orSEQ ID NO: 7.

The DNA hybridizable to the base sequence represented by SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 includes DNA containing abase sequence having at least about 70% homology, preferably at leastabout 80% homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology, to the base sequence representedby SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; etc.

The hybridization can be carried out by publicly known methods or bymodifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989), etc. A commercially available library may alsobe used according to the instructions of the attached manufacturer'sprotocol. More preferably, the hybridization can be carried out underhighly stringent conditions.

The highly stringent conditions used herein are, for example, those in asodium concentration at about 19 to 40 mM, preferably about 19 to 20 mMat a temperature of about 50 to 70° C., preferably about 60 to 65° C. Inparticular, hybridization conditions in a sodium concentration of about19 mM at a temperature of about 65° C. are most preferred.

More specifically, the DNA encoding GFP consisting of the amino acidsequence represented by SEQ ID NO: 1 includes DNA containing tie basesequence represented by SEQ ID NO: 2 (WO 97/42320), etc.; the DNAencoding GFP consisting of the amino acid sequence represented by SEQ IDNO: 3 includes DNA containing the base sequence represented by SEQ IDNO: 4 (WO 96/23810), etc.; the DNA encoding GFP (GFPuv) consisting ofthe amino acid sequence represented by SEQ ID NO: 5 includes DNAcontaining the base sequence represented SEQ ID NO: 6, etc.; the DNAencoding GFP (GFPuv) consisting of the amino acid sequence representedby SEQ ID NO: 5 includes DNA containing the base sequence representedSEQ ID NO: 6, etc. (WO 97/26333), etc.; the DNA encoding GFP (EGFP)consisting of the amino acid sequence represented by SEQ ID NO: 7includes DNA containing the base sequence represented SEQ ID NO: 8, etc.(NCBI Accession No. AAB0572), etc.

For the DNA encoding EGFP consisting of the amino acid sequencerepresented by SEQ ID NO: 7, there may be employed DNA consisting of thebase sequence represented by SEQ ID NO: 8, etc.

For the DNA encoding ECFP consisting of the amino acid sequencerepresented by SEQ ID NO: 22, there may be employed DNA consisting ofthe base sequence represented by SEQ ID NO: 23, etc.

For the DNA encoding EYFP consisting of the amino acid sequencerepresented by SEQ ID NO: 24, there may be employed DNA consisting ofthe base sequence represented by SEQ ID NO: 25, etc.

For the DNA encoding DsRED consisting of the amino acid sequencerepresented by SEQ ID NO: 26, there may be employed DNA consisting ofthe base sequence represented by SEQ ID NO: 27, etc.

For the DNA encoding EBFP consisting of the amino acid sequencerepresented by SEQ ID NO: 28, there may be employed DNA consisting ofthe base sequence represented by SEQ ID NO: 29, etc.

The fusion protein of an orphan receptor and a fluorescent protein canbe manufactured using DNA ligated with DNA encoding the orphan receptorprotein and DNA encoding GFP.

The ligated DNA is constructed in frame by ligating the 5′ end of DNAencoding GFP with the 3′ end of the base sequence of DNA encoding theorphan receptor.

Also, DNA encoding an amino acid sequence composed of approximately 1 to5 amino acid residues with a small molecular weight such as Ala, Gly,Ser, etc., which is called a linker, may be inserted between the bothDNAs.

The expression vector for the fusion protein of an orphan receptor and afluorescent protein (hereinafter sometimes simply referred to as thefusion protein) can be manufactured, for example, by (1) preparing a DNAfragment encoding the fusion protein and (2) ligating the DNA fragmentat the downstream of a promoter in an appropriate expression vector.

As the fusion protein of a receptor protein for which a ligand has notbeen identified and a fluorescent protein, preferably used are, forexample, fusion proteins of 102 kinds of receptor proteins for whichligands have not been identified, each receptor protein having the aminoacid sequence represented by any one of SEQ ID NO: 30 through SEQ ID NO:131, and GFP consisting of the amino acid sequence represented by SEQ IDNO: 1. In these amino acid sequences represented by any of SEQ ID NO: 30through SEQ ID NO: 131, the N-terminal methionine residue is deleted inthe amino acid sequence represented by SEQ ID NO: 1 and as the case maybe, the following alanine residue is further replaced by a threonineresidue or a serine residue.

The DNA encoding this fusion protein can be prepared by ligating DNA(SEQ ID NO: 2) encoding GFP consisting of the amino acid sequencerepresented by SEQ ID NO: 1 at the downstream of the region encodingeach of the 102 kinds of receptor proteins for which ligands have notbeen identified. Specifically, a DNA having the base sequencerepresented by either one of SEQ ID NO: 132 through SEQ ID NO: 233 isused. In these base sequences represented by any of SEQ ID NO: 132through SEQ ID NO: 233, the 5′ end methionine residue codon is deletedin the base sequence represented by SEQ ID NO: 2 and as the case may be,the following alanine residue codon is further replaced by a threonineresidue codon or a serine residue codon.

Examples of the expression vector include plasmids derived formEscherichia coli (e.g., pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13),plasmids derived from Bacillus subtilis (e.g., pUB 110, pTP5, pC194),plasmids derived from yeast (e.g., pSH19, pSH15), bacteriophages such asX phage, etc., animal viruses such as retrovirus, vaccinia virus,baculovirus, etc., as well as pA1-11, pXT1, pRc/CMV, pRc/RSV,pcDNAI/Neo, etc.

The promoter used in the vector may be any promoter if it is suitable tofunction well with a host used for gene expression. For example, in thecase of using animal cells as the host, SRα promoter, SV40 promoter, LTRpromoter, CMV promoter, HSV-TK promoter, etc. are used.

Further in the case of expressing in various tissues, there are employedinsulin II promoter (pancreas), Glycoprotein-α subunit promoter(pituitary), transthyretin promoter (liver), renin promoter (kidney),PSE promoter (prostate), CD2 promoter (T cell), IgG-heavy chain promoter(B cell), scavenger-receptor A promoter (macrophage), etc.

Where the host is bacteria of the genus Escherichia, preferred examplesof the promoter include trp promoter, lac promoter, recA promoter,λP_(L) promoter, 1pp promoter, etc. Where the host is bacteria of thegenus Bacillus as the host, preferred example of the promoter are SPO1promoter, SPO2 promoter, penP promoter, etc. When yeast is used as thehost, preferred examples of the promoter are PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, etc. When insect cells are used asthe host, preferred examples of the promoter include polyhedrinprompter, P10 promoter, etc.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a polyA additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV40ori) etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene(hereinafter sometimes abbreviated as Amp^(r)), neomycin resistant gene(hereinafter sometimes abbreviated as Neo^(r), G418 resistance), etc. Inparticular, when dhfr gene is used as the selection marker in CHO(dhfr⁻) cells, selection can also be made on thymidine free media.

If necessary, a signal sequence that matches with a host is added to theN-terminus of the receptor protein of the present invention. Examples ofthe signal sequence that can be used are Pho A signal sequence, OmpAsignal sequence, etc. in the case of using bacteria of the genusEscherichia as the host; α-amylase signal sequence, subtilisin signalsequence, etc. in the case of using bacteria of the genus Bacillus asthe host; MFα signal sequence, SUC2 signal sequence, etc. in the case ofusing yeast as the host; and insulin signal sequence, α-interferonsignal sequence, antibody molecule signal sequence, etc. in the case ofusing animal cells as the host, respectively.

Using the expression vector containing the DNA encoding the fusionprotein of the receptor protein and the fluorescent protein thusconstructed, transformants can be manufactured.

Examples of the host, which may be used, are bacteria belonging to thegenus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects, animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journalof Molecular Biology, 120, 517 (1978)), HB101 (Journal of MolecularBiology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)), DH5α (Inoue,H., Nojima, H., Gene, 96, 23-28 (1-990)), DH10B (Proc. Natl. Acad. Sci.USA, 87,4645-4649 (1990)), etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal ofBiochemistry, 95, 87 (1984)), etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cells (Sf cells), MG1 cells derived from mid-intestine ofTrichoplusia ni, High Five™ cells derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc. Where the virus is BmNPV, Bombyx mori N cells (BmN cells),etc. are used. Examples of the Sf cell which can be used are Sf9 cells(ATCC CRL1711), Sf21 cells (both cells are described in Vaughn, J. L. etal., In Vivo, 13, 213-217 (1977), etc.

As the insect, for example, a larva of Bombyx mori is used (Maeda, etal., Nature, 315, 592 (1985)).

Examples of animal cells, which are used, are monkey cells COS-7, Vero,Chinese hamster cells CHO (hereinafter referred to as CHO cells), dhfrgene deficient Chinese hamster cells CHO (hereinafter simply referred toas CHO (dhfr⁻) cell), mouse L cells, mouse AtT-20, mouse myeloma cells,rat GH3, human FL cells, pancreas-derived cells (RINm5F, HIT-T15, etc.),pituitary-derived cells (GH3, GH1, RC4/BC, etc.), placenta-derived cells(BeWo, JAR, JEG-3, etc.), liver-derived cells (HepG2, etc.),kidney-derived cells (ACHN, etc.), hematocyte-derived cells (H9, THP-1,U-937, etc.), HeLa cells, etc.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A., 69,2110 (1972), Gene, 17, 107 (1982) or the like.

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979), etc.

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55(1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995), published by Shujunsha, or Virology, 52, 456 (1973).

Thus, the transformant transformed with the expression vector containingthe DNA encoding the fusion protein described above can be obtained.

Where the host is bacteria belonging to the genus Escherichia or thegenus Bacillus, the transformant can be appropriately incubated in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, and so on. Examples of the carbon sources include glucose,dextrin, soluble starch, sucrose, etc. Examples of the nitrogen sourcesinclude inorganic or organic materials such as ammonium salts, nitratesalts, corn steep liquor, peptone, casein, meat extract, soybean cake,potato extract, etc. Examples of the inorganic materials are calciumchloride, sodium dihydrogenphosphate, magnesium chloride, etc. Inaddition, yeast extract, vitamins, growth promoting factors etc. mayalso be added to the medium. Preferably, pH of the medium is adjusted toabout 5 to about 8.

A preferred example of the medium for culturing the bacteria belongingto the genus Escherichia is M9 medium supplemented with glucose andCasamino acids [Miller, Journal of Experiments in Molecular Genetics,431-433, Cold Spring Harbor Laboratory, New York (1972)]. If necessary,a chemical such as 3β-indolylacrylic acid can be added to the mediumthereby to activate the promoter efficiently.

Where the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultured at about 15° C. to about 43°C. for about 3 hours to about 24 hours. If necessary, the culture may beaerated or stirred.

Where the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultured generally at about 30° C. to about 40° C.for about 6 hours to about 24 hours. If necessary, the culture can beaerated or stirred.

Where yeast is used as the host, the transformant is cultured, forexample, in Burkholder's minimal medium [Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in SD medium supplementedwith 0.5% Casamino acids [Bitter, G. A. et al., Proc. Nat]. Acad. Sci.U.S.A., 81, 5330 (1984)]. Preferably, pH of the medium is adjusted toabout 5 to about 8. In general, the transformant is cultured at about20° C. to about 35° C. for about 24 hours to about 72 hours. Ifnecessary, the culture can be aerated or stirred.

Where insect cells or insects are used as the host, the transformant iscultured in, for example, Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) to which an appropriate additive such asimmobilized 10% bovine serum is added. Preferably, pH of the medium isadjusted to about 6.2 to about 6.4. Normally, the transformant iscultured at about 27° C. for about 3 days to about 5 days and, ifnecessary, the culture can be aerated or stirred.

Where animal cells are employed as the host, the transformant iscultured in, for example, MEM medium containing about 5% to about 20%fetal calf serum [Science, 122, 501 (1952)), DMEM medium (Virology, 8,396 (1959)], RPMI 1640 medium [The Journal of the American MedicalAssociation, 199, 519 (1967)], 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultured at about 30° C. to about 40° C. for about 15 hours to about 60hours and, if necessary, the culture can be aerated or stirred.

Suitable media, conditions for culturing, etc. on the respective hostsare publicly known (especially see WO 00/14227, page 24, line 24 to page26, line 8, EP1111047A2, paragraphs [0090] to [0096]).

As above, the fusion protein can be expressed in the cell membrane ofthe transformant.

The fusion protein can be separated and purified from the culturedescribed above, for example, by the following procedures.

When the fusion protein is extracted from the culture or cells, afterculturing the transformants or cells are collected by a publicly knownmethod and suspended in an appropriate buffer. The transformants orcells are then disrupted by publicly known methods such asultrasonication, a treatment with lysozyme and/or freeze-thaw cycling,followed by centrifugation, filtration, etc. Thus, the crude extract ofthe fusion protein can be obtained. The buffer used for the proceduresmay contain a protein modifier such as urea or guanidine hydrochloride,or a surfactant such as Triton X-100™, etc. When the receptor protein issecreted in the culture, after completion of the cultivation thesupernatant can be separated from the transformants or cells to collectthe supernatant by a publicly known method.

The fusion protein contained in the supernatant or the extract thusobtained can be purified by appropriately combining the publicly knownmethods for separation and purification. Such publicly known methods forseparation and purification include a method utilizing difference insolubility such as salting out, solvent precipitation, etc.; a methodutilizing mainly difference in molecular weight such as dialysis,ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis,etc.; a method utilizing difference in electric charge such as ionexchange chromatography, etc.; a method utilizing difference in specificaffinity such as affinity chromatography, etc.; a method utilizingdifference in hydrophobicity such as reverse phase high performanceliquid chromatography, etc.; a method utilizing difference inisoelectric point such as isoelectrofocusing electrophoresis; and thelike.

When the fusion protein thus obtained is in a free form, it can beconverted into the salt by publicly known methods or modificationsthereof. On the other hand, when the fusion protein is obtained in theform of a salt, it can be converted into the free form or in the form ofa different salt by publicly known methods or modifications thereof.

The fusion protein produced by the recombinant can be treated, prior toor after the purification, with an appropriate protein modifying enzymeso that the fusion protein can be appropriately modified to partiallyremove a polypeptide. Examples of the protein-modifying enzyme includetrypsin, chymotrypsin, arginyl endopeptidase, protein kinase,glycosidase or the like.

The activity of the thus produced fusion protein or salts thereof can bedetermined by a test binding to a labeled ligand, by an enzymeimmunoassay using a specific antibody, or the like.

Examples of test compounds which are used for the ligand determinationmethod of the present invention include publicly known ligands (forexample, angiotensin, bombesin, canavinoid, cholecystokinin, glutamine,serotonin, melatonin, neuropeptide Y, opioids, purines, vasopressin,oxytocin, PACAP (e.g., PACAP27, PACAP38), secretin, glucagon,calcitonin, adrenomedulin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP(vasoactive intestinal and related polypeptide), somatostatin, dopamine,motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide),leukotriens, pancreastatin, prostaglandins, thromboxane, adenosine,adrenaline, the chemokine superfamily (e.g., the CXC chemokine subfamilysuch as IL-8, GROα, GROβ, GROγ, NAP-2, ENA-78, GCP-2, PF4, IP-10, Mig,PBSF/SDF-1, etc.; the CC chemokine subfamily such as MCAF/MCP-1, MCP-2,MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-1β, HCC-1, MIP-3α/LARC,MIP-3β/ELC, I-309, TARC, MIPF-1, MIPF-2/eotaxin-2, MDC, DC-CK1/PARC,SLC, etc.; the C chemokine subfamily such as lymphotactin, etc.; theCX3C chemokine subfamily such as fractalkine, etc.), endothelin,enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide,galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate,lysophosphatidylserine, sphingosylphosphorylcholine,lyzophosphatidylcholine, steroids, bile acids, isoprenoids, arachidonicacid metabolites, amines, amino acids, nucleotides, nucleosides,saturated or saturated fatty acids, etc.) as well as other substances,for example, tissue extracts, cell culture supernatants from humans ormammals (e.g., mice, rats, swine, bovine, sheep, monkeys, etc.). Forexample, the tissue extract, cell culture supernatant, etc. is added tothe cell wherein the fusion protein of the orphan receptor and GFP hasbeen expressed, and screening is made while assaying thecell-stimulating activities, etc. to finally determine a single ligand.

Specifically, the ligand determination method A of the present inventionis a method which involves constructing the fusion protein-expressedcell and assaying cell stimulating activities or perform a receptorbinding assay by using the expressed cell or its cell membrane fraction,thus determining compounds that bind to the orphan receptors to have thecell stimulating activities (e.g., activities that accelerate orsuppress arachidonic acid release, acetylcholine release, intracellularCa²⁺ release, intracellular cAMP production, intracellular cGMPproduction, inositol phosphate production, changes in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, etc.), MAP kinase activation, activation oftranscription factors (e.g., CRE, AP1, NFκB, etc.), diacylglycerolproduction, opening or closing of ion channels (e.g., K⁺, Ca²⁺, Na⁺,Cl⁻, etc.) on cell membranes, apoptosis induction, morphological changesin cells, transport of the receptor (fusion protein) from cell membraneto cytoplasm, activation of low molecular weight G proteins (e.g., Ras,Rap, Rho, Rab, etc.), cell division-promoting activity, DNAsynthesis-promoting activity, etc., that is, ligands (e.g., peptides,proteins, non-peptide compounds, synthetic compounds, fermentationproducts, etc.).

The ligand determination methods of the present invention arecharacterized by assaying, e.g., the binding amount of a test compoundto the orphan receptor or the cell stimulating activities, etc., when acell wherein the fusion protein has been expressed or a membranefraction of the cell is brought in contact with the test compound.

More specifically, the present invention provides the following liganddetermination methods:

-   -   (1) a method of determining a ligand to the orphan receptor,        which comprises assaying the amount of a labeled test compound        bound to a cell wherein the fusion protein has been expressed,        when the labeled test compound is brought in contact with the        cell or the cell membrane fraction;    -   (2) a method of determining a ligand to the orphan receptor,        which comprises assaying the amount of a labeled test compound        bound to a cell wherein the fusion protein has been expressed on        a cell membrane by culturing a transformant containing DNA        encoding the fusion protein, when the labeled test compound is        brought in contact with the fusion protein;    -   (3) a method of determining a ligand to the orphan receptor,        which comprises assaying the aforesaid cell stimulating        activities, MAP kinase activation, activation of transcription        factors (e.g., CRE, AP1, NFκB, etc.), diacylglycerol production,        opening or closing of ion channels (e.g., K⁺, Ca²⁺, Na⁺, Cl⁻,        etc.) on cell membranes, apoptosis induction, morphological        changes in cells, transport of the receptor (fusion protein)        from cell membrane to cytoplasm, activation of low molecular        weight G proteins (e.g., Ras, Rap, Rho, Rab, etc.), cell        division-promoting activity, DNA synthesis-promoting activity,        etc., which are mediated by the orphan receptor, when a test        compound is brought in contact with a cell wherein the fusion        protein has been expressed; and,    -   (4) a method of determining a ligand to the orphan receptor,        which comprises assaying the aforesaid mediated the cell        stimulating activities, MAP kinase activation, activation of        transcription factors (e.g., CRE, AP1, NFκB, etc.),        diacylglycerol production, opening or closing of ion channels        (e.g., K⁺, Ca²⁺, Na⁺, Cl⁻, etc.) on cell membranes, apoptosis        induction, morphological changes in cells, transport of the        receptor (fusion protein) from cell membrane to cytoplasm,        activation of low molecular weight G proteins (e.g., Ras, Rap,        Rho, Rab, etc.), cell division-promoting activity, DNA        synthesis-promoting activity, etc., which are mediated by the        orphan receptor, when a test compound is brought in contact with        the fusion protein expressed on a cell membrane by culturing a        transformant containing DNA encoding the fusion protein.

In particular, it is preferred to perform the tests (1) and (2)described above, thereby to confirm that the test compound can bind tothe orphan receptor, followed by the tests (3) and (4) described above.

As the case may be, the fusion protein can be isolated and purified fromthe expression cell described above and the receptor binding assay, etc.may be carried out using the same. For the fusion protein used in theligand determination method, fusion proteins abundantly expressed usingthe cells described above are appropriate.

The fusion protein can be manufactured by the method for expressiondescribed above, preferably by expressing DNA encoding the fusionprotein in mammalian or insect cells, etc. For introducing a DNAfragment encoding the fusion protein into host animal cells andefficiently expressing the same, it is preferred to insert the DNAfragment downstream a polyhedrin promoter of nuclear polyhedrosis virus(NPV) belonging to a baculovirus, an SV40-derived promoter, a retroviruspromoter, a metallothionein promoter, a human heat shock promoter, acytomegalovirus promoter, an SRa promoter or the like. The amount andquality of the fusion protein expressed can be determined by a publiclyknown method. For example, this determination can be made by the methoddescribed in the literature [Nambi, P., et al., J. Biol. Chem., 267,19555-19559 (1992)].

In the ligand determination methods of the present invention, it ispreferred to use the fusion protein-expressed cell or the cell membranefraction.

Where the fusion protein-expressed cells are used in the liganddetermination methods of the present invention, the cells may be fixedusing glutaraldehyde, formalin, etc. The fixation can be made bypublicly known methods.

The cells wherein the fusion protein has been expressed refer to hostcells wherein the fusion protein has been expressed, which host cellsinclude Escherichia coli, Bacillus subtilis, yeast, insect cells, animalcells, and the like.

The cell membrane fraction means a fraction abundant in cell membraneobtained by cell disruption and subsequent fractionation by publiclyknown methods. Useful cell disruption methods include cell squashingusing a Potter-Elvehjem homogenizer, disruption using a Waring blenderor Polytron (manufactured by Kinematica Inc.), disruption byultrasonication, disruption by cell spraying through thin nozzles underan increased pressure using a French press, or the like. Cell membranefractionation is effected mainly by fractionation using a centrifugalforce, such as centrifugation for fractionation and density gradientcentrifugation. For example, cell disruption fluid is centrifuged at alow speed (500 rpm to 3,000 rpm) for a short period of time (normallyabout 1 to about 10 minutes), the resulting supernatant is thencentrifuged at a higher speed (15,000 rpm to 30,000 rpm) normally for 30minutes to 2 hours. The precipitate thus obtained is used as themembrane fraction. The membrane fraction is rich in the fusion proteinexpressed and membrane components such as cell-derived phospholipids,membrane proteins, etc.

The amount of the fusion protein in the fusion protein-expressed cellsor the cell membrane fraction is preferably 10³ to 10⁸ molecules percell, more preferably 10⁵ to 10⁷ molecules per cell. As the amount ofexpression increases, the ligand binding activity per unit of membranefraction (specific activity) increases so that not only the highlysensitive screening system can be constructed but also large quantitiesof samples can be assayed with the same lot. The amount of this fusionprotein expressed can be roughly estimated from the fluorescence levelof GFP in the cells or cell membranes, using a fluorescence microscopeor a fluorophotometer.

To perform the methods (1) and (2) described above for determining aligand to the orphan receptor, appropriate cells or cell membranefractions containing the fusion protein and a labeled test compound arerequired. The fusion protein fraction is desirably a fraction containinga recombinant fusion receptor having an activity equivalent to anaturally occurring fusion protein. Herein, the term “oequivalentactivity” means a ligand binding activity, a signal transductionactivity, etc., which is equivalent.

As the labeled test compounds, there are used compounds selected fromthe aforesaid group of ligand compounds, which are labeled with [³H],[¹²⁵I], [¹⁴C], [³⁵S], etc.

Specifically, the ligand determination methods of the present inventioncan be performed by the following procedures. First, a standard fusionprotein preparation is prepared by suspending cells wherein the fusionprotein has been expressed or the cell membrane fraction in a bufferappropriate for use in the determination method. Any buffer can be usedso long as it does not inhibit the ligand-receptor binding, such buffersincluding a phosphate buffer or a Tris-HCl buffer having pH of 4 to 10(preferably pH of 6 to 8). For the purpose of minimizing non-specificbinding, a surfactant such as CHAPS, Tween-80™ (manufactured byKao-Atlas Inc.), digitonin or deoxycholate, and various proteins such asbovine serum albumin or gelatin, may optionally be added to the buffer.Further for the purpose of suppressing the degradation of the receptorsor ligands by proteases, a protease inhibitor such as PMSF, leupeptin,E-64 (manufactured by Peptide Institute, Inc.) and pepstatin may also beadded. A given amount (5,000 to 500,000 cpm) of the test compoundlabeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S] or the like is added to 0.01 mlto 10 ml of the receptor preparation. To determine the amount ofnon-specific binding (NSB), a reaction tube containing an unlabeled testcompound in a large excess is also prepared. The reaction is carried outat approximately 0 to 50° C., preferably about 4 to 37° C. for about 20minutes to about 24 hours, preferably about 30 minutes to about 3 hours.After completion of the reaction, the reaction mixture is filtratedthrough glass fiber filter paper, etc. and washed with an appropriatevolume of the same buffer. The residual radioactivity on the glass fiberfilter paper is then measured by means of a liquid scintillation counteror γ-counter. A test compound exceeding 0 cpm in count obtained bysubtracting nonspecific binding (NSB) from the total binding (B) (Bminus NSB) may be selected as a ligand (including an agonist) to theorphan receptor.

The method (3) or (4) of the present invention described above fordetermining a ligand can be performed as follows. The aforesaid orphanreceptor-mediated cell stimulating activities, for example, MAP kinaseactivation, activation of transcription factors (e.g., CRE, AP1, NFκB,etc.), diacylglycerol production, opening or closing of ion channels(e.g., K⁺, Ca²⁺, Na⁺, Cl⁻, etc.) on cell membranes, apoptosis induction,morphological changes in cells, transport of the receptor (fusionprotein) from cell membrane into cytoplasm, activation of low molecularweight G proteins (e.g., Ras, Rap, Rho, Rab, etc.), celldivision-promoting activity, DNA synthesis-promoting activity, etc. canbe assayed by publicly known methods or using assay kits commerciallyavailable. Specifically, cells wherein the fusion protein has expressedare first cultured on a multi-well plate, etc. Prior to the liganddetermination, the medium is replaced with fresh medium or with anappropriate non-cytotoxic buffer, followed by incubation for a givenperiod of time in the presence of a test compound, etc. Subsequently,the cells are extracted or the supernatant is recovered and theresulting product is quantified by appropriate procedures. Where it isdifficult to detect the production of the indicator substance (e.g.,arachidonic acid, etc.) for the cell-stimulating activity due to adegrading enzyme contained in the cells, an inhibitor against such adegrading enzyme may be added prior to the assay. For detectingactivities such as the cAMP production suppression activity, thebaseline production in the cells is increased by forskolin or the likeand the suppressing effect on the increased baseline production may thenbe detected.

In the cell stimulating activities described above, “transport of thereceptor to cytoplasm” is assayed by measuring the fluorescence of afluorescent protein such as GFP, etc., whereby movement of the fusionprotein from the cell membrane to the cytoplasm can be observed. Inorder to observe the movement of the fusion protein from the cellmembrane to the cytoplasm, it is suited to use animal cells which haveexpressed the aforesaid fusion protein stably or transiently. The cellsare incubated in an appropriate incubator using an ordinary medium and atest compound diluted to a suitable concentration is added to the cells.In this case, a diluted solution of the test compound may be addeddirectly to the medium, or after the cells are washed with Hanks'balanced salt solution (HBSS, to which BSA may be added in 0 to 10%,preferably 0.1 to 1%), HBSS containing the test compound may be added tothe cells. The cells are allowed to stand at 4° C. to 37° C., preferablyat 20° C. to 37° C. for 1 minute to 6 hours, preferably for 10 minutesto 2 hours, followed by observing movement of the fusion protein fromthe cell membrane to the cytoplasm. Though the cells can be observed asthey are, the cells may also be fixed with glutaraldehyde, formalin,etc. The cells can be fixed by publicly known methods.

Observation may be made using an ordinary fluorescence microscope orconfocal laser scanning microscope. Also, a plate reader having thefunction to irradiate excitement light and the function to takefluorescent images in can be used. In this case, after excitement withultraviolet light, preferably at 395 nm, the fusion protein of thereceptor and wild type GFP or GFPuv represented by SEQ ID NO: 3 or SEQID NO: 5 can be detected using fluorescein isothianate (FITC) or filtersnormally commercially available for detecting GFP. In order to detectthe fusion protein with GFP represented by SEQ ID NO: 1 or SEQ ID NO: 7,excitement may be effected with excitement light at 460-500 nm,preferably at 488 nm and observation may be made using FITC or filtersnormally commercially available for detecting GFP.

In addition to the transport of the receptor (fusion protein) intocytoplasm, morphological changes of the receptor including receptoraggregation, receptor localization, decreased or increased expression ofthe receptor, etc., which can be microscopically observed, may also beobserved.

In practicing the ligand determination method B of the presentinvention, it is necessary to incorporate a plasmid containing a DNAencoding the reporter protein at the downstream of a particularenhancer/promoter into cells, preferably eukaryote-derived cells, inaddition to the expression vector for the fusion protein. This plasmidmay contain a promoter capable of expressing the reporter protein incells, preferably in eukaryote-derived cells, may further contain achemical resistant gene (e.g., ampicillin-resistant gene), etc. as aselection marker in the case of proliferating in eukaryotes.

The plasmid containing a DNA encoding the reporter protein at thedownstream of the enhancer/promoter may be any plasmid such ascommercially available plasmid, etc., so far as it is capable ofexpressing the reporter protein in cells under control of the enhancerand is capable of introducing into cells.

As the enhancer, there is used, for example, a virus-derived enhancersuch as papilloma virus, etc., LTR of a retrovirus, cAMP responseelement (CRE), TPA response element (TRE) or the like, preferably cAMPresponse element. An enhancer which can be activated by the aforesaidcell stimulating activities mediated by the orphan receptor the cellsexpress is appropriate.

As the promoter, there is used, for example, SV40 promoter, a TATA-likepromoter of HSV thymidine kinase gene, etc., with a TATA-like promoterbeing preferred.

As the reporter protein gene, there is used, for example, luciferasegene, β-galactosidase gene, GFP gene, alkaline phosphatase gene, etc.Any enzyme gene is usable as the reporter gene so long as its enzymeactivity can be detected by publicly known methods.

Specific examples of such plasmids include a plasmid ligated with aTATA-like promoter and a reporter protein (e.g., luciferase gene) at thedownstream of cAMP response element, e.g., pCRE-Luc (Clontech, Inc.),etc.

The cells used in the ligand determination method B are the host cellsdescribed above, preferably animal cells (e.g., monkey cells COS-7,Vero, CHO cells, CHO (dhfr⁻) cells, mouse L cells, mouse AtT-20, mousemyeloma cells, rat GH3, human FL cells, human HEK293 cells, etc.) andthe like.

The cells may express 2 or more (preferably 2 or 3) fusion proteins.

Where 2 or more fusion proteins are expressed, it is preferred to use 2or more orphan receptors having similar biological characteristics.

Examples of the similar biological characteristics include expressionlevels of the reporter protein when 2 or more orphan receptors used areexpressed independently; etc. Specifically, the characteristics of therespective receptor proteins can be distinguished, using as an indicator(1) the basic expression level of the reporter protein and/or (2) theexpression level of the reporter protein in the presence of forskolin,when 2 or more orphan receptors are expressed individually.

Accordingly, when 2 or more orphan receptors are expressed to determinethe ligand, it is desired to previously sort the reporter proteins intothose having low, medium and obviously high basic expression levels,etc., or to clarify the receptor protein difficult to increase anexpression level of the reporter protein by the addition of forskolin.This is because where two receptor protein, for example, a receptorprotein having a high basic expression level of the reporter protein anda receptor having a low basic expression level of the reporter proteinare expressed, it becomes difficult to detect an increase in theexpression level of the reporter protein when a ligand binds to thelatter. That is:

-   -   (1) it is preferred to avoid the mixture of an orphan receptor        that the basic expression level of the reporter protein is high        and a receptor protein that the basic expression level is low;    -   (2) it is preferred not to mix a receptor protein that the        expression level of the reporter protein increased by the        addition of forskolin is remarkably high with an orphan receptor        that the expression level increased is not; and,    -   (3) it is preferred that orphan receptors having almost equal        basic expression levels of the reporter protein are used and        expressed in combination.

As such, the combination of orphan receptors having similarcharacteristics includes, for example, the combination of APJ (apelinreceptor; Gene, 136, 355 (1993)) and TGR-1 (Japanese Patent ApplicationKOKAI No. 2002-078492).

The ligand determination method B is specifically described below.

Cells are plated on a 96-well plate and incubated overnight in DMEMcontaining, e.g., 10% fetal calf serum. At this stage, an expressionplasmid and a reporter plasmid in the fluorescent protein areco-transfected to the cells, using a transfection kit commerciallyavailable. The cells are further incubated overnight to express theorphan receptor transiently in the cells. After the cells are washed andthe medium is made serum-free, a test compound is added. Where theenhancer is CRE, forskolin may be added simultaneously with the testcompound. After incubation is carried out for a given period of time,the cells are lysed and the activities of the reporter protein areassayed.

In the determination method described above, when the base line for theactivities of the reporter protein is high so that a change in theactivities is detected by a test compound only with difficulties, it ispreferred to take measures for reducing the base line. For example inthe case that the orphan receptor is a G protein-coupled receptorprotein (GPCR), Gi protein, among a subunits of G protein, showing acAMP inhibitory effect is added, which makes the detection of a changein activities easy. To express Gi protein, a plasmid capable ofexpressing a DNA encoding Gi protein can be transfected to cellstogether with the orphan receptor plasmid and the reporter plasmid. Inthis case, a mixing ratio of these three plasmids (the orphan receptorplasmid, the reporter plasmid and the Gi plasmid) are preferably inabout 5-15:1:1 to 6, more preferably in about 7:1:3.

In the ligand determination method B of the present invention, where theactivities of the reporter protein increase or decrease by about 20% ormore, preferably by about 50% or more when a test compound is added, thetest compound can be identified to be a ligand.

The test compound used in the ligand determination method B of thepresent invention is a compound selected from the test compoundsdescribed above.

Furthermore, the kit of the present invention for determination of theligand contains cells capable of expressing the fusion protein of thepresent invention or its cell membrane fraction, etc.

Examples of the ligand determination kit of the present invention aregiven below.

1. Reagents for Determining Ligands

(1) Assay Buffer and Wash Buffer

Hanks' Balanced Salt Solution (manufactured by Gibco Co.) supplementedwith 0.05% bovine serum albumin (Sigma Co.).

The solution is sterilized by filtration through a 0.45 μm filter andstored at 4° C. Alternatively, the solution may be prepared at use.

(2) Fusion Protein Preparation

CHO cells wherein the fusion protein has been expressed are passaged ina 12-well plate in a density of 5×10⁵ cells/well followed by culturingat 37° C. under 5% CO₂ and 95% air for 2 days.

(3) Labeled Test Compound

Compounds labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc., which arecommercially available labels, or compounds labeled by appropriatemethods.

An aqueous solution of the compound is stored at 4° C. or −20° C. Thesolution is diluted to 1 μM with an assay buffer at use. A sparinglywater-soluble test compound is dissolved in dimethylformamide, DMSO,methanol, etc.

(4) Non-Labeled Test Compound

A non-labeled form of the same compound as the labeled compound isprepared in a concentration of 100 to 1,000-fold higher than that of thelabeled compound.

2. Assay Method

-   -   (1) CHO cells wherein the fusion protein has been expressed are        cultured in a 12-well culture plate. After washing twice with 1        ml of an assay buffer, 490 μl of the assay buffer is added to        each well.    -   (2) After 5 μl of the labeled test compound is added, the        resulting mixture is incubated at room temperature for an hour.        To determine the non-specific binding, 5 μl of the non-labeled        compound is added to the system.    -   (3) The reaction mixture is removed and the wells are washed 3        times with 1 ml of washing buffer. The labeled test compound        bound to the cells is dissolved in 0.2N NaOH-1% SDS and then        mixed with 4 ml of liquid scintillator A (manufactured by Wako        Pure Chemical Industries, Ltd.).    -   (4) The radioactivity is measured using a liquid scintillation        counter (manufactured by Beckman Co.).

As such, according to the ligand determination methods (A and B) of thepresent invention, immunostaining or western blot assay utilizing thefluorescence from the fluorescent protein such as GFP, etc. or afluorescent protein antibody such as a GFP antibody, etc. is used toeffect (1) to (5) below:

-   -   (1) it can be confirmed that the receptor protein has been        expressed on a protein level;    -   (2) it can be confirmed that the receptor protein has been        expressed on a cell membrane;    -   (3) an expression level. of the receptor protein can be        estimated;    -   (4) a cell wherein the receptor protein has been expressed        abundantly can be selected; and,    -   (5) a specific reaction of the receptor by a ligand can be        detected as internalization of the fusion protein of the        receptor and the fluorescent protein into cells. By utilizing        these features, a ligand to the receptor protein for which a        ligand has not been identified (orphan receptor) can be        determined efficiently.

The ligand thus determined binds to its receptor protein to regulate itsphysiological functions and thus can be used as an agent for theprevention and/or treatment of diseases associated with the functions ofthe receptor protein. Further using the ligand and its receptor protein,an agonist/antagonist to the receptor can be screened.

In the specification and drawings, the codes of bases and amino acidsare denoted in accordance with the IUPAC-IUB Commission on BiochemicalNomenclature or by the common codes in the art, examples of which areshown below. For amino acids that may have the optical isomer, L form ispresented unless otherwise indicated.

The sequence identification numbers in the sequence listing of thespecification indicates the following sequence, respectively.

[SEQ ID NO: 1]

This shows the amino acid sequence of GFP used in EXAMPLE 1 (hereinaftermerely referred to as GFP-1).

SEQ ID NO: 2

This shows the base sequence of DNA encoding GFP used in EXAMPLE 1.

SEQ ID NO: 3

This shows the amino acid sequence of wild type GFP.

SEQ ID NO: 4

This shows the base sequence of DNA encoding wild type GFP.

SEQ ID NO: 5

This shows the amino acid sequence of GFPuv.

SEQ ID NO: 6

This shows the base sequence of DNA encoding GFPuv.

SEQ ID NO: 7

This shows the amino acid sequence of EGFP.

SEQ ID NO: 8

This shows the base sequence of cDNA encoding EGFP.

SEQ ID NO: 9

This shows the amino acid sequence of human-derived G protein-coupledreceptor protein TGR5 used in EXAMPLE 1.

SEQ ID NO: 10

This shows the base sequence of cDNA encoding human-derived Gprotein-coupled receptor protein TGR5 used in EXAMPLE 1.

SEQ ID NO: 11

This shows the base sequence of primer 1 used for PCR in REFERENCEEXAMPLE 1.

SEQ ID NO: 12

This shows the base sequence of primer 2 used for PCR in REFERENCEEXAMPLE 1.

SEQ IDNO: 13

This shows the amino acid sequence of human-derived parathyroid hormonereceptor (PTH-R).

SEQ ID NO: 14

This shows the base sequence of cDNA encoding human-derived parathyroidhormone receptor (PTH-R).

SEQ ID NO: 15

This shows the amino acid sequence of human-derived GPR40.

SEQ ID NO: 16

This shows the base sequence of cDNA encoding human-derived GPR40.

SEQ ID NO: 17

This shows the amino acid sequence of His-Tag.

SEQ ID NO: 18

This shows the amino acid sequence of V5-tag.

SEQ ID NO: 19

This shows the amino acid sequence of myc-tag.

SEQ ID NO: 20

This shows the amino acid sequence of Xpress-tag.

SEQ ID NO: 21

This shows the amino acid sequence of HA-tag.

SEQ ID NO: 22

This shows the amino acid sequence of ECFP.

SEQ ID NO: 23

This shows the base sequence of cDNA encoding ECFP.

SEQ ID NO: 24

This shows the amino acid sequence of EYFP.

SEQ ID NO: 25

This shows the base sequence of cDNA encoding EYFP.

SEQ ID NO: 26

This shows the amino acid sequence of DsRED.

SEQ ID NO: 27

This shows the base sequence of cDNA encoding DsRED.

SEQ ID NO: 28

This shows the amino acid sequence of EBFP.

SEQ ID NO: 29

This shows the base sequence of cDNA encoding EBFP.

SEQ ID NO: 30

This shows the amino acid sequence of the fusion protein of orphanreceptor hBL5 and GFP-1.

SEQ ID NO: 31

This shows the amino acid sequence of the fusion protein of orphanreceptor h7TBA62 and GFP-1.

SEQ ID NO: 32

This shows the amino acid sequence of the fusion protein of orphanreceptor 14273 and GFP-1.

SEQ ID NO: 33

This shows the amino acid sequence of the fusion protein of orphanreceptor EMR3 and GFP-1.

SEQ ID NO: 34

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR15 and GFP-1.

SEQ ID NO: 35

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR31 and GFP-1.

SEQ ID NO: 36

This shows the amino acid sequence of the fusion protein of orphanreceptor GPRC5B and GFP-1.

SEQ ID NO: 37

This shows the amino acid sequence of the fusion protein of orphanreceptor PSEC0142 and GFP-1.

SEQ ID NO: 38

This shows the amino acid sequence of the fusion protein of orphanreceptor HE6 and GFP-1.

SEQ ID NO: 39

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR61 and GFP-1.

SEQ ID NO: 40

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR9 and GFP-1.

SEQ ID NO: 41

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR24 and GFP-1.

SEQ ID NO: 42

This shows the amino acid sequence of the fusion protein of orphanreceptor ZGPR1 and GFP-1.

SEQ ID NO: 43

This shows the amino acid sequence of the fusion protein of orphanreceptor EMR1 and GFP-1.

SEQ ID NO: 44

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR25 and GFP-1.

SEQ ID NO: 45

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR55 and GFP-1.

SEQ ID NO: 46

This shows the amino acid sequence of the fusion protein of orphanreceptor AXOR14 and GFP-1.

SEQ ID NO: 47

This shows the amino acid sequence of the fusion protein of orphanreceptor TM7SF1 and GFP-1.

SEQ ID NO: 48

This shows the amino acid sequence of the fusion protein of orphanreceptor PSP24B and GFP-1.

SEQ ID NO: 49

This shows the amino acid sequence of the fusion protein of orphanreceptor SREB3 and GFP-1.

SEQ ID NO: 50

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR37 and GFP-1.

SEQ ID NO: 51

This shows the amino acid sequence of the fusion protein of orphanreceptor H963 and GFP-1.

SEQ ID NO: 52

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR87 and GFP-1.

SEQ ID NO: 53

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR91 and GFP-1.

SEQ ID NO: 54

This shows the amino acid sequence of the fusion protein of orphanreceptor PNR and GFP-1.

SEQ ID NO: 55

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR29 and GFP-1.

SEQ ID NO: 56

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR36 and GFP-1.

SEQ ID NO: 57

This shows the amino acid sequence of the fusion protein of orphanreceptor H9 and GFP-1.

SEQ ID NO: 58

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR18 and GFP-1.

SEQ ID NO: 59

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR19 and GFP-1.

SEQ ID NO: 60

This shows the amino acid sequence of the fusion protein of orphanreceptor AM-R and GFP-1.

SEQ ID NO: 61

This shows the amino acid sequence of the fusion protein of orphanreceptor GPRI9 and GFP-1:

SEQ ID NO: 62

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR45 and GFP-1.

SEQ ID NO: 63

This shows the amino acid sequence of the fusion protein of orphanreceptor GPRC5D and GFP-1.

SEQ ID NO: 64

This shows the amino acid sequence of the fusion protein of orphanreceptor LGR6 and GFP-1.

SEQ ID NO: 65

This shows the amino acid sequence of the fusion protein of orphanreceptor RUP3 and GFP-1.

SEQ ID NO: 66

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR14 and GFP-1.

SEQ ID NO: 67

This shows the amino acid sequence of the fusion protein of orphanreceptor TPRA40 and GFP-1.

SEQ ID NO: 68

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR22 and GFP-1.

SEQ ID NO: 69

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR52 and GFP-1.

SEQ ID NO: 70

This shows the amino acid sequence of the fusion protein of orphanreceptor FLH2882 and GFP-1.

SEQ ID NO: 71

This shows the amino acid sequence of the fusion protein of orphanreceptor SNORF36 and GFP-1.

SEQ ID NO: 72

This shows the amino acid sequence of the fusion protein of orphanreceptor MRG and GFP-1.

SEQ ID NO: 73

This shows the amino acid sequence of the fusion protein of orphanreceptor SREB2 and GFP-1.

SEQ ID NO: 74

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR12 and GFP-1.

SEQ ID NO: 75

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR30 and GFP-1.

SEQ ID NO: 76

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR82 and GFP-1.

SEQ ID NO: 77

This shows the amino acid sequence of the fusion protein of orphanreceptor RECAP and GFP-1.

SEQ ID NO: 78

This shows the amino acid sequence of the fusion protein of orphanreceptor HB954 and GFP-1.

SEQ ID NO: 79

This shows the amino acid sequence of the fusion protein of orphanreceptor RDC 1 and GFP-1.

SEQ ID NO: 80

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR6 and GFP-1.

SEQ ID NO: 81

This shows the amino acid sequence of the fusion protein of orphanreceptor A-2 and GFP-1.

SEQ ID NO: 82

This shows the amino acid sequence of the fusion protein of orphanreceptor JEG1 8 and GFP-1.

SEQ ID NO: 83

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR17 and GFP-1.

SEQ ID NO: 84

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR35 and GFP-1.

SEQ ID NO: 85

This shows the amino acid sequence of the fusion protein of orphanreceptor GPRC5C and GFP-1.

SEQ ID NO: 86

This shows the amino acid sequence of the fusion protein of orphanreceptor HM74 and GFP-1.

SEQ ID NO: 87

This shows the amino acid sequence of the fusion protein of orphanreceptor RPE and GFP-1.

SEQ ID NO: 88

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR13 and GFP-1.

SEQ ID NO: 89

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR27 and GFP-1.

SEQ ID NO: 90

This shows the amino acid sequence of the fusion protein of orphanreceptor DEZ and GFP-1.

SEQ ID NO: 91

This shows the amino acid sequence of the fusion protein of orphanreceptor ratGPR1 and GFP-1.

SEQ ID NO: 92

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR3 and GFP-1.

SEQ ID NO: 93

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR6 and GFP-1.

SEQ ID NO: 94

This shows the amino acid sequence of the fusion protein of orphanreceptor RAIG1 and GFP-1.

SEQ ID NO: 95

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR2-1 and GFP-1.

SEQ ID NO: 96

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR2-2 and GFP-1.

SEQ ID NO: 97

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR21 and GFP-1.

SEQ ID NO: 98

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR56 and GFP-1.

SEQ ID NO: 99

This shows the amino acid sequence of the fusion protein of orphanreceptor KIAA0758 and GFP-1.

SEQ ID NO: 100

This shows the amino acid sequence of the fusion protein of orphanreceptor RE2 and GFP-1.

SEQ ID NO: 101

This shows the amino acid sequence of the fusion protein of orphanreceptor P40 and GFP-1.

SEQ ID NO: 102

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR27 and GFP-1.

SEQ ID NO: 103

This shows the amino acid sequence of the fusion protein of orphanreceptor HG38 and GFP-1.

SEQ ID NO: 104

This shows the amino acid sequence of the fusion protein of orphanreceptor DRR1 and GFP-1.

SEQ ID NO: 105

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR12 and GFP-1.

SEQ ID NO: 106

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR11 and GFP-1.

SEQ ID NO: 107

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR15 and GFP-1.

SEQ ID NO: 108

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR8 and GFP-1.

SEQ ID NO: 109

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR20 and GFP-1.

SEQ ID NO: 110

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR10 and GFP-1.

SEQ ID NO: 111

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR30 and GFP-1.

SEQ ID NO: 112

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR18 and GFP-1.

SEQ ID NO: 113

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR25 and GFP-1.

SEQ ID NO: 114

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR23 and GFP-1.

SEQ ID NO: 115

This shows the amino acid sequence of the fusion protein of orphanreceptor P2Y10 and GFP-1.

SEQ ID NO: 116

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR37 and GFP-1.

SEQ ID NO: 117

This shows the amino acid sequence of the fusion protein of orphanreceptor ET(B)R-LP-2 and GFP-1.

SEQ ID NO: 118

This shows the amino acid sequence of the fusion protein of orphanreceptor FPRL2 and GFP-1.

SEQ ID NO: 119

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR32 and GFP-1.

SEQ ID NO: 120

This shows the amino acid sequence of the fusion protein of orphanreceptor dj287G14.2 and GFP-1.

SEQ ID NO: 121

This shows the amino acid sequence of the fusion protein of orphanreceptor BRS-3 and GFP-1.

SEQ ID NO: 122

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR39 and GFP-1.

SEQ ID NO: 123

This shows the amino acid sequence of the fusion protein of orphanreceptor 63A2 and GFP-1.

SEQ ID NO: 124

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR84 and GFP-1.

SEQ ID NO: 125

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR21 and GFP-1.

SEQ ID NO: 126

This shows the amino acid sequence of the fusion protein of orphanreceptor GPR48 and GFP-1.

SEQ ID NO: 127

This shows the amino acid sequence of the fusion protein of orphanreceptor SNORF1 and GFP-1.

SEQ ID NO: 128

This shows the amino acid sequence of the fusion protein of orphanreceptor BA12 and GFP-1.

SEQ ID NO: 129

This shows the amino acid sequence of the fusion protein of orphanreceptor MAS and GFP-1.

SEQ ID NO: 130

This shows the amino acid sequence of the fusion protein of orphanreceptor OT7T009 and GFP-1.

SEQ ID NO: 131

This shows the amino acid sequence of the fusion protein of orphanreceptor TGR34 and GFP-1.

SEQ ID NO: 132

This shows the base sequence of DNA encoding the fusion protein oforphan receptor hBL5 and GFP-1.

SEQ ID NO: 133

This shows the base sequence of DNA encoding the fusion protein oforphan receptor h7TBA62 and GFP-1.

SEQ ID NO: 134

This shows the base sequence of DNA encoding the fusion protein oforphan receptor 14273 and GFP-1.

SEQ ID NO: 135

This shows the base sequence of DNA encoding the fusion protein oforphan receptor EMR3 and GFP-1.

SEQ ID NO: 136

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR15 and GFP-1.

SEQ ID NO: 137

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR31 and GFP-1.

SEQ ID NO: 138

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPRC5B and GFP-1.

SEQ ID NO: 139

This shows the base sequence of DNA encoding the fusion protein oforphan receptor PSEC0142 and GFP-1.

SEQ ID NO: 140

This shows the base sequence of DNA encoding the fusion protein oforphan receptor HE6 and GFP-1.

SEQ ID NO: 141

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR61 and GFP-1.

SEQ ID NO: 142

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR9 and GFP-1.

SEQ ID NO: 143

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR24 and GFP-1.

SEQ ID NO: 144

This shows the base sequence of DNA encoding the fusion protein oforphan receptor ZGPR1 and GFP-1.

SEQ ID NO: 145

This shows the base sequence of DNA encoding the fusion protein oforphan receptor EMR1 and GFP-1.

SEQ ID NO: 146

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR25 and GFP-1.

SEQ ID NO: 147

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR55 and GFP-1.

SEQ ID NO: 148

This shows the base sequence of DNA encoding the fusion protein oforphan receptor AXOR14 and GFP-1.

SEQ ID NO: 149

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TM7SF1 and GFP-1.

SEQ ID NO: 150

This shows the base sequence of DNA encoding the fusion protein oforphan receptor PSP24B and GFP-1.

SEQ ID NO: 151

This shows the base sequence of DNA encoding the fusion protein oforphan receptor SREB3 and GFP-1.

SEQ ID NO: 152

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR37 and GFP-1.

SEQ ID NO: 153

This shows the base sequence of DNA encoding the fusion protein oforphan receptor H963 and GFP-1.

SEQ ID NO: 154

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR87 and GFP-1.

SEQ ID NO: 155

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR91 and GFP-1.

SEQ ID NO: 156

This shows the base sequence of DNA encoding the fusion protein oforphan receptor PNR and GFP-1.

SEQ ID NO: 157

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR29 and GFP-1.

SEQ ID NO: 158

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR3 6 and GFP-1.

SEQ ID NO: 159

This shows the base sequence of DNA encoding the fusion protein oforphan receptor H9 and GFP-1.

SEQ ID NO: 160

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR18 and GFP-1.

SEQ ID NO: 161

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR19 and GFP-1.

SEQ ID NO: 162

This shows the base sequence of DNA encoding the fusion protein oforphan receptor AM-R and GFP-1.

SEQ ID NO: 163

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR19 and GFP-1.

SEQ ID NO: 164

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR45 and GFP-1.

SEQ ID NO: 165

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPRC5D and GFP-1.

SEQ ID NO: 166

This shows the base sequence of DNA encoding the fusion protein oforphan receptor LGR6 and GFP-1.

SEQ ID NO: 167

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RUP3 and GFP-1.

SEQ ID NO: 168

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR14 and GFP-1.

SEQ ID NO: 169

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TPRA40 and GFP-1.

SEQ ID NO: 170

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR22 and GFP-1.

SEQ ID NO: 171

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR52 and GFP-1.

SEQ ID NO: 172

This shows the base sequence of DNA encoding the fusion protein oforphan receptor FLH2882 and GFP-1.

SEQ ID NO: 173

This shows the base sequence of DNA encoding the fusion protein oforphan receptor SNORF36 and GFP-1.

SEQ ID NO: 174

This shows the base sequence of DNA encoding the fusion protein oforphan receptor MRG and GFP-1.

SEQ ID NO: 175

This shows the base sequence of DNA encoding the fusion protein oforphan receptor SREB2 and GFP-1.

SEQ ID NO: 176

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR12 and GFP-1.

SEQ ID NO: 177

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR30 and GFP-1.

SEQ ID NO: 178

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR82 and GFP-1.

SEQ ID NO: 179

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RECAP and GFP-1.

SEQ ID NO: 180

This shows the base sequence of DNA encoding the fusion protein oforphan receptor HB954 and GFP-1.

SEQ ID NO: 181

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RDC1 and GFP-1.

SEQ ID NO: 182

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR6 and GFP-1.

SEQ ID NO: 183

This shows the base sequence of DNA encoding the fusion protein oforphan receptor A-2 and GFP-1.

SEQ ID NO: 184

This shows the base sequence of DNA encoding the fusion protein oforphan receptor JEG18 and GFP-1.

SEQ ID NO: 185

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR17 and GFP-1.

SEQ ID NO: 186

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR35 and GFP-1.

SEQ ID NO: 187

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPRC5C and GFP-1.

SEQ ID NO: 188

This shows the base sequence of DNA encoding the fusion protein oforphan receptor HM74 and GFP-1.

SEQ ID NO: 189

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RPE and GFP-1.

SEQ ID NO: 190

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR 13 and GFP-1.

SEQ ID NO: 191

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR27 and GFP-1.

SEQ ID NO: 192

This shows the base sequence of DNA encoding the fusion protein oforphan receptor DEZ and GFP-1.

SEQ ID NO: 193

This shows the base sequence of DNA encoding the fusion protein oforphan receptor ratGPR1 and GFP-1.

SEQ ID NO: 194

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR3 and GFP-1.

SEQ ID NO: 195

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR6 and GFP-1.

SEQ ID NO: 196

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RAIG1 and GFP-1.

SEQ ID NO: 197

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR2-1 and GFP-1.

SEQ ID NO: 198

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR2-2 and GFP-1.

SEQ ID NO: 199

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR21 and GFP-1.

SEQ ID NO: 200

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR56 and GFP-1.

SEQ ID NO: 201

This shows the base sequence of DNA encoding the fusion protein oforphan receptor KIAA0758 and GFP-1.

SEQ ID NO: 202

This shows the base sequence of DNA encoding the fusion protein oforphan receptor RE2 and GFP-1.

SEQ ID NO: 203

This shows the base sequence of DNA encoding the fusion protein oforphan receptor P40 and GFP-1.

SEQ ID NO: 204

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR27 and GFP-1.

SEQ ID NO: 205

This shows the base sequence of DNA encoding the fusion protein oforphan receptor HG38 and GFP-1.

SEQ ID NO: 206

This shows the base sequence of DNA encoding the fusion protein oforphan receptor DRR1 and GFP-1.

SEQ ID NO: 207

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR12 and GFP-1.

SEQ ID NO: 208

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR11 and GFP-1.

SEQ ID NO: 209

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR15 and GFP-1.

SEQ ID NO: 210

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR8 and GFP-1.

SEQ ID NO: 211

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR20 and GFP-1.

SEQ ID NO: 212

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR10 and GFP-1.

SEQ ID NO: 213

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR30 and GFP-1.

SEQ ID NO: 214

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR1 8 and GFP-1.

SEQ ID NO: 215

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR25 and GFP-1.

SEQ ID NO: 216

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR23 and GFP-1.

SEQ ID NO: 217

This shows the base sequence of DNA encoding the fusion protein oforphan receptor P2Y10 and GFP-1.

SEQ ID NO: 218

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR37 and GFP-1.

SEQ ID NO: 219

This shows the base sequence of DNA encoding the fusion protein oforphan receptor ET(B)R-LP-2 and GFP-1.

SEQ ID NO: 220

This shows the base sequence of DNA encoding the fusion protein oforphan receptor FPRL2 and GFP-1.

SEQ ID NO: 221

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR32 and GFP-1.

SEQ ID NO: 222

This shows the base sequence of DNA encoding the fusion protein oforphan receptor dj287G14.2 and GFP-1.

SEQ ID NO: 223

This shows the base sequence of DNA encoding the fusion protein oforphan receptor BRS-3 and GFP-1.

SEQ ID NO: 224

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR39 and GFP-1.

SEQ ID NO: 225

This shows the base sequence of DNA encoding the fusion protein oforphan receptor 63A2 and GFP-1.

SEQ ID NO: 226

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR84 and GFP-1.

SEQ ID NO: 227

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR21 and GFP-1.

SEQ ID NO: 228

This shows the base sequence of DNA encoding the fusion protein oforphan receptor GPR48 and GFP-1.

SEQ ID NO: 229

This shows the base sequence of DNA encoding the fusion protein oforphan receptor SNORF1 and GFP-1.

SEQ ID NO: 230

This shows the base sequence of DNA encoding the fusion protein oforphan receptor BA12 and GFP-1.

SEQ ID NO: 231

This shows the base sequence of DNA encoding the fusion protein oforphan receptor MAS and GFP-1.

SEQ ID NO: 232

This shows the base sequence of DNA encoding the fusion protein oforphan receptor OT7T009 and GFP-1.

SEQ ID NO: 233

This shows the base sequence of DNA encoding the fusion protein oforphan receptor TGR34 and GFP-1.

The transformant Escherichia coli JM109/pCR4-hTGR5 obtained in REFERENCEEXAMPLE 1 has been on deposit with International Patent OrganismsDepository, National Institute of Advanced Industrial Science andTechnology (formerly, National Institute of Bioscience andHuman-Technology (NIBH), Ministry of Economics, Trade and Industry)located at Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki (post code:305-8566), as the Accession Number FERM BP-7114 since Apr. 3, 2000, andwith Institute for Fermentation (IFO), located at 2-17-85 Juso-honmachi,Yodogawa-ku, Osaka-shi, Osaka (post code: 532-8686), as the AccessionNumber IFO 16410 since March 23, 2000.

EXAMPLES

The present invention is described in detail below with reference toEXAMPLES and REFERENCE EXAMPLES, but is not deemed to limit the scope ofthe present invention thereto. The gene manipulation procedures usingEscherichia coli were performed according to the methods described inthe Molecular Cloning.

Reference Example 1

Cloning of cDNA Encoding G Protein-Coupled Receptor Protein of HumanSpleen and Determination of the Base Sequence

Using human spleen-derived cDNA (Clontech) as a template and twoprimers, namely, primer 1 (SEQ ID NO: 11) and primer 2 (SEQ ID NO: 12),PCR was carried out. The reaction solution in the above reactioncomprised of 1/10 volume of the cDNA described above as a template, 1/50volume of Advantage-GC2 Polymerase Mix (Clontech), 0.5 μM each of primer1 (SEQ ID NO: 11) and primer 2 (SEQ ID NO: 12), 200 μM of dNTPs, 1/5volume of a buffer attached to the enzyme product and 1/5 volume of GCMelt to make the total volume 20 11. The PCR reaction was carried out byreacting at 94° C. for 5 minutes, then repeating 30 times a cycle set toinclude 94° C. for 30 seconds, 60° C. for 30 seconds and 68° C. for 2minutes, and finally conducting extension at 68° C. for 5 minutes. ThePCR product was subcloned to plasmid vector pCR4 (Invitrogen) followingthe instructions attached to the TA Cloning Kit (Invitrogen). Theplasmid was then transfected to Escherichia coli JM109, and the clonescontaining the cDNA of the PCR product were selected on LB agar platescontaining ampicillin. As a result of analysis for the sequence of eachclone, cDNA sequence encoding a novel G protein-coupled receptor proteinwas obtained (SEQ ID NO: 10). The novel G protein-coupled receptorprotein containing the amino acid sequence (SEQ ID NO: 9) deduced fromthis cDNA sequence was named TGR5. Also, the transformant containing DNArepresented by SEQ ID NO: 10 was named Escherichia coliJM109/pCR4-hTGR5.

Reference Example 2

Detection of Reporter Activation with Cholesterol Metabolism-RelatedSubstance in Human HEK293 Cells wherein TGR5 was Expressed Transiently

The TGR5-specific stimulation activity by cholesterol metabolism-relatedsubstance was detected using as an indicator the expression level of areporter gene product (luciferase) produced by expression induction ofCRE promoter.

Human-derived HEK293 cells were suspended in growth redium (DMEM(Dulbecco's Modified Eagle Medium) (Gibco BRL) supplemented with 10%fetal cow serum (Gibco BRL)). The suspension adjusted to a concentrationof 1×10⁵ cells/well was plated on a collagen-coated black well 96-wellplate (Becton Dickinson, Inc.).

After culturing overnight at 37° C. under the 5% CO₂ condition, the TGR5gene was inserted into an expression vector for animal cell pAKKO-111H(the same plasmid vector as pAKKO-1.111H described in Biochem. Biophys.Acta, Hinuma, S. et al., 1219, 251-259, 1994) by publicly known methods,together with a reporter gene-containing plasmid pCRE-Luc (Clontech).The thus prepared TGR5 expression vector plasmid or intact pAKKO-111Hcontaining no TGR5 gene was transfected to the cells as described below.

By mixing OPTI-MEM-I (Gibco BRL, Inc.) and Lipofectamine™ 2000 reagent(Gibco BRL, Inc.) in 24:1, a Lipofectamine dilution was prepared. Also,OPTI-MEM-I, TGR5 expression vector plasmid or the intact vector plasmid(240 ng/μl) and pCRE-Luc (240 ng/μl) were mixed in 24:0.9:0.1 to preparea DNA dilution. The Lipofectamine dilution and the DNA dilution weremixed in equal volumes and the mixture was allowed to stand for 20minutes at room temperature to produce the DNA-Lipofectamine complex.Then, 25 μl of the solution was added to the HEK293 cell-incubated platedescribed above, followed by further culturing overnight at 37° C. underthe 5% CO₂ condition.

The transfected HEK293 cells were washed with an assay medium (DMEMsupplemented with 0.1% bovine serum albumin) and lithocholic acid (WakoPure Chemical Industries, Inc.) and progesterone (Wako Pure ChemicalIndustries, Inc.) diluted with the assay medium to 2×10⁻⁵ M were addedto the plate, which was then incubated for 4 hours at 37° C. under the5% CO₂ condition. The culture supernatant was discarded and 50 μl of asubstrate for assaying the luciferase activity or PicaGene LT 2.0 (ToyoInk Mfg. Co., Ltd.) was added. The fluorescence level of luciferase wasmeasured by using a plate reader (ARVO sx multi-label counter, Wallac,Inc.).

As a result, an increased luciferase activity by lithocholic acid andprogesterone was noted specifically to the HEK293 cells transfected withTGR5 gene having the base sequence represented by SEQ ID NO: 10 (FIG.1).

Reference Example 3

Transfection of G Protein-Coupled Receptor Protein-Expressed Plasmid andReporter Plasmid to Host Cells

Using expression plasmids for animal cells inserted with various Gprotein-coupled receptor protein cDNAs prepared by publicly knownmethods, i.e., thyrotropin releasing hormnone receptor (TRHR),neuromedin U receptor (FM-3 and TGR-1), prolactin releasing factorreceptor (hGR3), apelin receptor (APJ), etc., Escherichia coli JM109 wastransfected and the colonies obtained were isolated and cultured.Thereafter, plasmid was prepared using QIAGEN Plasmid Maxi Kit (Qiagen).Furthermore, the reporter plasmid of pCRE-Luc (Clontech) ligated with aluciferase gene as a reporter at the downstream of cAMP response element(CRE) was prepared in a similar manner.

Human HEK293 cells as host cells for transfecting the G protein-coupledreceptor protein and the reporter plasmid thereto were plated on a typeI collagen-coated 96-well black plate (Becton Dickinson, Inc.) in100,000 cells/well in a culture solution volume of 100 μl, followed byculturing overnight. In the same way, CHO (dhfr-) cells were transformedby pAKKO-111H and the resulting CHO-mock cells were plated on a 96-wellblack plate of Costar, Inc. in 40,000 cells/well in a culture solutionvolume of 100 μl, followed by culturing overnight. In the both cells,DMEM (Gibco BRL, Inc.) supplemented with 10% fetal calf serum alone wasused as a medium for plate culture.

Each of the plasmids above was diluted to a concentration of 240 ng/μl,and the dilution was added to 240 μl of Opti-MEM-I (Gibco BRL, Inc.) ina proportion of 9 μl of the G protein-coupled receptor proteinexpression plasmid and 1 μl of the reporter plasmid. The mixture wasmixed with 240 μl of Opti-MEM-I added with 10 μl of Lipofectamine 2000(Gibco BRL, Inc.) in equal volumes to form the liposome and plasmidcomplex, in accordance with the procedures in the instruction manualsattached to Lipofectamine 2000. Also, for carrying out efficientscreening, 5 μl each of the 3 receptor expression plasmids was added ina concentration of 240 ng/μl and the mixtures having the same proportionof the other reagents as described above were prepared. The mixtureswere added to the culture solution of HEK293 or CHO-mock cells in 25 μleach/well and incubated at 37° C. overnight to introduce the plasmids.In the CHO-mock cells, the culture solution was replaced by an assaybuffer (DMEM supplemented with 0.1% bovine serum albumin) 4 hours afterthe plasmid addition to make serum-free.

Reference Example 4

Detection of Ligand Activity by Reporter Assay

In the HEK293 cells, the culture solution was replaced by the assaybuffer of REFERENCE EXAMPLE 3 an hour before the assaying andpre-incubated. A solution of a ligand or a ligand candidate compound inthe assay buffer was prepared and added to the HEK293 cells or CHO-mockcells prepared in REFERENCE EXAMPLE 3. Also, an assay was conducted in asimilar manner under the conditions where forskolin was added to theassay buffer in the final concentration of 2 μM. After the ligand or thetest compound was added, incubation was carried out for 4 hours toinduce promotion or suppression of the transcription/translation of thereporter gene derived from intracellular signal transduction caused bythe agonist activity of ligand mediated by the receptor. After theincubation was completed, the assay buffer in each well was removed and50 μl each of luminescent substrate PicaGene LT 2.0 (Toyo Ink Mfg. Co.,Ltd.) was added to the well. After the cells were lysed and thoroughlymixed with the substrate, the luminescent level corresponding to theexpression induction level of the reporter gene in each well wasmeasured on the plate reader described in REFERENCE EXAMPLE 2.

Using expression plasmids in which various G protein-coupled receptorprotein cDNAs were inserted in accordance with the procedures describedin REFERENCE EXAMPLES 3 and 4, the expression induction of the reportergene by ligand stimulation was assayed in HEK293 cells. In CRFR coupledto GS as a subunit of G protein for transduction of signals into cells,activation of the reporter gene due to the ligand addition was detectedunder both conditions in the absence of and in the presence offorskolin. In APJ coupled to inhibitory Gi, suppressed expression of thereporter gene due to the ligand addition was detected under thecondition that forskolin was added. Further in the receptors TRHR, FM-3and TGR-1 coupled to Gq, promoted expression of the reporter gene wasdetected under the condition that forskolin was added. Also in thereceptor hGR3 coupled to both Gq and Gi, promoted expression of thereporter gene was detected as well, under the condition that forskolinwas added (FIG. 2).

Reference Example 5

Reporter Assay using Inhibitory G Protein a Subunit Gi-Expressed Plasmid

Inhibitory G protein α subunit (Gi)-expressed plasmid was prepared in asimilar manner to the G protein receptor-expressed plasmid shown inREFERENCE EXAMPLE 3 (herein Gi may be any kind irrespective of anyanimal species). This plasmid, the receptor-expressed plasmid and thereporter plasmid were added to 240 μl of Opti-MEM-I in the proportion of3 μl, 7 μl and 1 μl and DNA was introduced into HEK293 or CHO-mock cellsotherwise under the same conditions as in EXAMPLE 2. The mixing ratio ofthese 3 plasmids is from 1 to 6 μl of Gi, preferably from 1 to 3 μl,when the total amount is made 11 μl. These plasmids were assayed inaccordance with the procedures of EXAMPLE 3 and the ligand activitieswere detected.

That is, the response of TGR5 to lithocholic acid in the co-presence ofGi was detected. As a result, Gi was co-expressed together with TGR5 inthe assay for G protein receptor TGR5 using the CHO-mock cells, wherebythe luciferase activity added with no ligand (ligand (−)) could bemarkedly reduced, which enabled to detect the increase in activity bythe ligand (lithocholic acid, 2×10⁻⁵ M, ligand (+)) (FIG. 3).

Example 1

Internalization of the TGR5-GFP Fusion Protein Expressed on CHO Cells bythe Addition of Taurolithocholic Acid

An expression plasmid was constructed to express the fusion protein inwhich TGR5 was fused at the C terminus with Green Fluorescent Protein(GFP) isolated from jelly fish Aequorea victoria. In this case, afragment excised from expression vector pQBI25 (TaKaRa Shuzo) for GFPwas used as cDNA (SEQ ID NO: 2) of GFP. In the cDNA of TGR5, itstermination codon was corrected by PCR to the recognition sequence ofrestriction enzyme NheI, and the cDNA fragment of GFP was ligatedthereto, which was inserted into expression vector pAKKO-111H describedin EXAMPLE 1. The expression vector plasmid for the thus obtained fusionprotein of TGR5 and GFP (hereinafter TGR5-GFP) was transfected toCHO-mock cells by the following procedures. The CHO-mock cells weresuspended in growth medium [DMEM (Dulbecco's Modified Eagle Medium)(GIBCO BRL, Inc.) supplemented with 10% fetal calf serum (GIBCO BRL,Inc.)] and plated on a Lab-TekII coverglass chamber (Nalgen Nunc, Inc.)with 4 chamber compartments in a concentration of 0.6×10⁵ cells/chamber.After culturing overnight at 37° C. under the 5% CO₂ condition,transfection was effected. For the transfection, Lipofectamine™ 2000reagent (GIBCO BRL, Inc.) was used. First, 2 μl of Lipofectamine™ 2000reagent was mixed with 50 μl of OPTI-MEM-I (GIBCO BRL, Inc.). Afterallowing to stand for 5 minutes, the mixture wasgmixed with a solutionmixture of 0.48 μg of DNA and 50 μl of OPTI-MEM-1. The mixture was leftto stand at room temperature for 20 minutes, thereby to form theDNA-lipofectamine complex. After 100 μl of the mixture was added to thechamber above where the CHO cells were incubated, incubation wasconducted overnight at 37° C. under the 5% CO₂ condition. The medium wasreplaced by medium for confocal laser scanning microscopic observation[suspension of 0.1% bovine albumin (Essentially Fatty Acid Free (GIBCOBRL, Inc.) in Hanks' Balanced Salt Solution (GIBCO BRL, Inc.))] and thefluorescent image of GFP was observed on a confocal laser scanningmicroscope (Leica). In this case, GFP was excited at 488 nm.

As a result, the TGR5-GFP fluorescent protein was observed in the cellmembrane (FIG. 4). Taurolithocholic acid was added to the cells to have10⁻⁵ M in the medium. It was found that 30 minutes after, thefluorescence of GFP was moved to the cytoplasm, not in the cell membrane(FIG. 5). This indicates that TGR5 is a G protein-coupled receptor andat the same time, TGR5 moves to the cytoplasm, namely, internalized, inresponse to taurolithocholic acid.

Example 2

Expression of the Fusion Protein of Parathyroid Hormone Receptor (PTH-R)and GFP in Pancreas β Cell Line RINm5F by Transient Expression

Expression vector was prepared in a manner similar to EXAMPLE 1 byinserting an expression plasmid for expressing the fusion protein ofhuman PTH-R (SEQ ID NO: 13) and GFP at the C terminus into expressionvector pAKKO-111H described in REFERENCE EXAMPLE 2. The expressionvector plasmid for the thus obtained fusion protein of PTH-R and GFP(hereinafter PTH-GFP) was transfected to RINm5F cells by the followingprocedures. The RINm5F cells were suspended in growth medium [RPMI1640(GIBCO BRL, Inc.) supplemented with 10% fetal calf serum (GIBCO BRL,Inc.) treated by Charcoal/Dextran] and plated on a Lab-TekII coverglasschamber (Nalgen Nunc, Inc.) with 8 chamber compartments in aconcentration of 0.3×10⁵ cells/chamber. After culturing overnight at 37°C. under the 5% CO₂ condition, transfection was effected. For thetransfection, Lipofectamine™ 2000 reagent (GIBCO BRL, Inc.) was used.First, 3.3 μl of Lipofectamine™ 2000 reagent was mixed with 80 μl ofOPTI-MEM-I (GIBCO BRL, Inc.). After allowing to stand for 5 minutes, themixture was mixed with a solution mixture of 0.72 Hg of DNA and 80 μl ofOPTI-MEM-I. The mixture was left to stand at room temperature for 20minutes, thereby to form the DNA-lipofectamine complex. After 160 μl ofthe solution mixture was added to the chamber above where the RINm5Fcells were incubated, the cells were incubated overnight at 37° C. underthe 5% CO₂ condition. Confocal laser scanning microscopic observationwas performed in a manner similar to EXAMPLE 1.

As a result, it was observed that the PTH-GFP fusion protein wasexpressed in the cell membrane.

Example 3

Expression of the GPCR and GFP Fusion Protein using Insulin II Promoter

In a manner similar to EXAMPLE 1, a fragment of human GPR40 (SEQ ID NO:15) ligated with GFP at the C terminus was inserted at the downstream ofmouse insulin II promoter cloned from mouse genome to prepare anexpression vector for expressing the fusion protein of GPR40 and GFP(hereinafter GPR40-GFP). The expression vector plasmid for the thusobtained GPR40-GFP was transfected to MIN6 cells by the followingprocedures. The MIN6 cells were suspended in growth medium [DMEM(containing 4.5 g/l Glucose) (Invitrogen, Inc.) supplemented with 15%fetal calf serum (Trace, Inc.) in the final concentration, 55 μM of2-mercaptoethanol (Invitrogen, Inc.) and 20 mM HEPES (DainipponPharmaceutical Co., Ltd.)] and plated on a Lab-TekII coverglass chamber(Nalgen Nunc, Inc.) with 4 chamber compartments in a concentration of1.2×10⁵ cells/chamber. After culturing for 2 nights at 37° C. under the5% CO₂ condition, transfection was effected. For the transfection,Lipofectamine™ 2000 reagent (GIBCO BRL, Inc.) was used. First, 4 μl ofLipofectamine™ 2000 reagent was mixed with 100 μl of Opti-MEM(Invitrogen, Inc.). After allowing to stand for 5 minutes, the mixturewas mixed with a solution mixture of 2 μg of DNA and 100 μl of Opti-MEMmedium. The mixture was left to stand at room temperature for 20minutes, thereby to form the DNA-lipofectamine complex. After 100 μl ofthe solution mixture was added to the chamber above where the MIN6 cellswere incubated, the cells were incubated for 4 hours at 37° C. under the5% CO₂ condition. Then, the medium was replaced by 400 μl of a freshgrowth medium followed by further incubation overnight. Confocal laserscanning microscopic observation was made as in EXAMPLE 1.

As a result, the GPR40-GFP fluorescent protein was observed in the cellmembrane of MIN6 cells.

Example 4

Preparation of Transformants Capable of Individually Expressing 102Kinds of Fusion Proteins

Expression vector plasmids of 102 kinds containing the respective DNAs(SEQ ID NO: 132 through SEQ ID NO: 233) encoding the fusion proteins of102 kinds of the respective receptor proteins for which ligands have notbeen identified and GFP consisting of the amino acid sequencerepresented by SEQ ID NO: 1 or its modified amino acid sequences wereprepared, and transfected into CHO-mock cells. These CHO cells wereincubated as in EXAMPLE 1. It was observed that the fusion proteins wereexpressed in the CHO-mock cells.

Industrial Applicability

Since various cell lines can be used, the methods of the presentinvention for determining ligands to receptor proteins for which ligandshave not been identified are simple and can be performed in a shortperiod of time.

1. A method of determining a ligand to a receptor protein for which aligand has not been identified, which comprises using a fusion proteinof the receptor protein and a fluorescent protein.
 2. The liganddetermination method according to claim 1, wherein the fusion protein ofa receptor protein for which a ligand has not been identifyd and GFP isused.
 3. The ligand determination method according to claim 1, wherein acell expressed with the fusion protein of a receptor protein for which aligand has not been identified and GFP or a membrane fraction of thecell is brought in contact with a test compound.
 4. The liganddetermination method according to claim 1, which comprises assaying (1)an activity that accelerates or suppresses arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos or pH reduction, (2) MAPkinase activation, (3) transcription factor activation, (4)diacylglycerol production, (5) opening or closing of ion channels on acell membrane, (6) apoptosis induction, (7) morphological changes incells, (8) transport of the fusion protein from cell membrane tocytoplasm, (9) low molecular weight G protein activation, (10) celldivision-promoting activity or (11) DNA synthesis-promoting activity. 5.The ligand determination method according to claim 1, wherein transportof the fusion protein from cell membrane to cytoplasm is assayed.
 6. Theligand determination method according to claim 5, wherein transport ofthe fusion protein from cell membrane to cytoplasm is assayed byobserving the fluorescence of GFP.
 7. The ligand determination methodaccording to claim 1, which comprises bringing a cell capable ofexpressing the fusion protein of a receptor protein for which a ligandhas not been identified the receptor protein and a fluorescent proteinand containing a plasmid ligated with a DNA encoding a reporter proteinat the downstream of cAMP response element/promoter in contact with atest compound and assaying the activity of the reporter protein.
 8. Themethod according to claim 7, which comprises culturing a cell containing(1) a plasmid containing a DNA encoding the fusion protein of a receptorprotein for which a ligand has not been identified and a fluorescentprotein and (2) a plasmid ligated with a DNA encoding a reporter proteinat the downstream of cAMP response element/promoter, and bringing thecell in contact with a test compound and assaying the activity of thereporter protein.
 9. The ligand determination method according to claim2, wherein a cell capable of expressing the fusion protein of a receptorprotein for which a ligand has not been identified and GFP andcontaining a plasmid ligated with a DNA encoding a reporter protein atthe downstream of cAMP response element/promoter is brought in contactwith a test compound and assaying the activity of the reporter protein.10. The ligand determination method according to claim 9, whichcomprises culturing a cell containing (1) a plasmid containing a DNAencoding the fusion protein of a receptor protein for which a ligand hasnot been identified and GFP and (2) a plasmid ligated with a DNAencoding a reporter protein at the downstream of cAMP responseelement/promoter, bringing the cell in contact with a test compound andassaying the activity of the reporter protein.
 11. The method accordingto claim 1, wherein the receptor protein is a G protein-coupled receptorprotein.
 12. The method according to claim 1, wherein GFP is a proteincontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5 or SEQ ID NO:
 7. 13. The method according to claim 7, wherein thepromoter is a TATA-like sequence.
 14. The method according to claim 7,wherein the reporter protein is luciferase.
 15. The method according toclaim 7, wherein the plasmid is a plasmid ligated with a TATA-likepromoter and a gene encoding the reporter protein at the downstream ofcAMP response element.
 16. The method according to claim 7, wherein thecell has expressed at least two receptor proteins for which ligands havenot been identified.
 17. The method according to claim 7, wherein thecell further contains a plasmid containing a gene encoding an inhibitoryG protein a subunit Gi.
 18. The method according to claim 7, whereinforskolin is further added.
 19. The method according to claim 16,wherein the at least two receptor proteins have similar characteristics.20. The method according to claim 19, wherein the similarcharacteristics are the basic expression level of the reporter proteinand/or the expression level of the reporter protein when fdrskolin isadded.
 21. The method according to claim 16, which comprises measuringthe basic expression level of the reporter protein when the at least tworeceptor proteins are expressed individually and/or the expression levelof the reporter protein by the addition of forskolin, and beingexpressed in combination the at leas two receptor proteins having anequivalent expression level of the reporter protein.
 22. A fusionprotein of a receptor protein for which a ligand has not been identifiedand a fluorescent protein, or a salt thereof.
 23. The fluorescentprotein or a salt thereof according to claim 22, wherein the fluorescentprotein is GFP.
 24. A DNA containing a DNA encoding the fusion proteinaccording to claim
 22. 25. A recombinant vector containing the DNAaccording to claim
 22. 26. A transfornant transformed with therecombinant vector according to claim
 25. 27. Use of a fluorescentprotein to determine a ligand to a receptor protein for which a ligandhas not been identified.
 28. Use of GFP to determine a ligand to areceptor protein for which a ligand has not been identified.